Progress toward re-engineering non-ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

Stevens, Brian W.; Joska, Tammy M.; Anderson, Amy C.

doi:10.1002/ddr.20041

Cited by 15 publications

(15 citation statements)

References 40 publications

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“…The primary substrate-specifying role of the A domain has made this domain a key target for NRPS recombination experiments. However, efforts to generate novel peptide products by modifying A domain substrate specificity, or by substituting in a non-synonymous A domain (that is, one which specifies an alternative monomer to the A domain it is replacing) have generally been unsuccessful [ 7 , 8 ]. Numerous in vitro e.g., [ 9 , 10 ] and in vivo [ 11 , 12 ] studies have demonstrated that the C domain typically “proof-reads” the downstream acceptor substrate (Fig.…”

Section: Introductionmentioning

confidence: 99%

Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases

Calcott

Ackerley

2015

BMC Microbiol

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BackgroundNon-ribosomal peptide synthetase (NRPS) enzymes govern the assembly of amino acids and related monomers into peptide-like natural products. A key goal of the field is to develop methods to effective recombine NRPS domains or modules, and thereby generate modified or entirely novel products. We previously showed that substitution of the condensation (C) and adenylation (A) domains in module 2 of the pyoverdine synthetase PvdD from Pseudomonas aeruginosa led to synthesis of modified pyoverdines in a minority of cases, but that more often the recombinant enzymes were non-functional. One possible explanation was that the majority of introduced C domains were unable to effectively communicate with the thiolation (T) domain immediately upstream, in the first module of PvdD.ResultsTo test this we first compared the effectiveness of C-A domain substitution relative to T-C-A domain substitution using three different paired sets of domains. Having previously demonstrated that the PvdD A/T domain interfaces are tolerant of domain substitution, we hypothesised that T-C-A domain substitution would lead to more functional recombinant enzymes, by maintaining native T/C domain interactions. Although we successfully generated two recombinant pyoverdines, having a serine or a N5-formyl-N5-hydroxyornithine residue in place of the terminal threonine of wild type pyoverdine, in neither case did the T-C-A domain substitution strategy lead to substantially higher product yield. To more comprehensively examine the abilities of non-native T domains to communicate effectively with the C domain of PvdD module 2 we then substituted the module 1 T domain with 18 different T domains sourced from other pyoverdine NRPS enzymes. In 15/18 cases the recombinant NRPS was functional, including 6/6 cases where the introduced T domain was located upstream of a C domain in its native context.ConclusionsOur data indicate that T domains are generally able to interact effectively with non-native C domains, contrasting with previous findings that they are not generally portable upstream of epimerisation (E) or thioesterase (TE) domains. This offers promise for NRPS recombination efforts, but also raises the possibility that some C domains are unable to efficiently accept non-native peptides at their donor site due to steric constraints or other limitations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0496-3) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases

Calcott

Ackerley

2015

BMC Microbiol

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“…Non-ribosomal peptides (NRPs) are structurally complex natural products and potentially important antiinfective agents generated by non-ribosomal peptide synthetases (NRPSs), a family of large and modular multi-enzymes (Stevens et al 2005). In general, each module of NRPSs is composed of three core domains, condensation (C), adenylation (A), and peptidyl carrier protein (PCP) and the A-domain recruits a specific amino acid substrate to be incorporated into NRP structures (Marahiel et al 1997;Schwarzer et al 2003).…”

Section: Introductionmentioning

confidence: 99%

Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs

et al. 2012

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Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit L: -Tyr, L: -Val, L: -Ile, L: -allo-Ile, or L: -Phe, which diversifies the fusaricidin structures. Since the L: -Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate L: -Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain.

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“…Previous efforts to generate novel peptide products in vivo by NRPS manipulation have involved either (i) replacement of the A domain by one that activates an alternative substrate, (ii) alteration of the substrate binding pocket of the A domain, or (iii) substitutions that treat C-A domains as inseparable pairs (8). However, in nearly all cases, these manipulations have resulted in little to no product being made.…”

mentioning

confidence: 99%

Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosa

Calcott

Owen

Lamont

et al. 2014

Appl Environ Microbiol

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dPyoverdine is a fluorescent nonribosomal peptide siderophore made by fluorescent pseudomonads. The Pseudomonas aeruginosa nonribosomal peptide synthetase (NRPS) PvdD contains two modules that each incorporate an L-threonine residue at the C-terminal end of pyoverdine. In an attempt to generate modified pyoverdine peptides, we substituted alternative-substratespecifying adenylation (A) and peptide bond-catalyzing condensation (C) domains into the second module of PvdD. When just the A domain was substituted, the resulting strains produced only wild-type pyoverdine-at high levels if the introduced A domain specified threonine or at trace levels otherwise. The high levels of pyoverdine synthesis observed whenever the introduced A domain specified threonine indicated that these nonnative A domains were able to communicate effectively with the PvdD C domain. Moreover, the unexpected observation that non-threonine-specifying A domains nevertheless incorporated threonine into pyoverdine suggests that the native PvdD C domain exhibited stronger selectivity than these A domains for the incorporated amino acid substrate (i.e., misactivation of a threonine residue by the introduced A domains was more frequent than misincorporation of a nonthreonine residue by the PvdD C domain). In contrast, substitution of both the C and A domains of PvdD generated high yields of rationally modified pyoverdines in two instances, these pyoverdines having either a lysine or a serine residue in place of the terminal threonine. However, C-A domain substitution more commonly yielded a truncated peptide product, likely due to stalling of synthesis on a nonfunctional recombinant NRPS template.

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Progress toward re-engineering non-ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

Cited by 15 publications

References 40 publications

Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases

Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases

Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs

Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosa

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